Application of DESs in extraction or in-situ separation of statins components in red yeast rice

Abstract

The invention discloses an application of DESs in extraction or in-situ separation of statins components in red yeast rice. The DESs are Btacc / Eg, Btacc / Gly, Btacc / Dim, Tebrc / La or Tebrc / Dim. According to the invention, the use of an organic solvent is avoided, operation units are reduced, and the method has great application potential; compared with a traditional organic solvent, the enzyme system is more compatible with cellulase and has higher enzyme catalysis efficiency; the strong solubility of DESs on a substrate can overcome the limitation of poor solubility of a traditional water-enzyme system; according to the present invention, the optimal process for extracting the effective components in the red yeast rice based on the BTAC / Eg (1: 2) DESs system is obtained, under the optimal extraction condition, the total extraction rate of the effective components is 9.209 mg / g, the extraction efficiency is higher than the extraction efficiency of the conventional solvent, and the extraction and the separation of the effective components in the red yeast rice are easily promoted.

Description

Technical Field

[0001] The invention relates to the application of DESs in extracting or in-situ separating statin components in red yeast rice, and belongs to the application field of low eutectic solvents. Background Art

[0002] As a new generation of green solvents, deep eutectic solvents (DESs) have attracted widespread attention due to their excellent physical and chemical properties. They are non-flammable, thermally stable, low toxicity, simple preparation, and low raw material prices, and have shown great potential in many fields. Compared with water and traditional organic solvents, DESs have stronger solubility for hydrophilic and hydrophobic compounds. In addition, DESs also show good biocompatibility. DESs are a low eutectic mixture composed of a certain stoichiometric ratio of hydrogen-bond donors (HBD) and hydrogen-bond acceptors (HBA).

[0003] After natural products are extracted, a mixed solution containing the target compound is obtained, which includes active ingredients, inactive ingredients and impurities. The content of the target compound in the extract is relatively low. In order to increase its content in the extract, it is necessary to further remove impurities and other components. Traditional separation methods are generally independent of extraction, in-situ separation and other operations, and require additional solvents. Not only does it consume a large amount of organic solvents, it is time-consuming and labor-intensive, but it is also inefficient, which greatly limits the development and application of natural products. Studies have shown that DESs with polyalkyl-substituted ammonium chloride as hydrogen bond acceptors and polyols and acids as hydrogen bond donors have pH-responsive phase behavior, and the conversion between the two phases can be achieved by adjusting the pH. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide the application of DESs in the extraction, in-situ separation or recovery of statins in red yeast rice, which helps to promote the extraction and separation of active ingredients in red yeast rice, avoids the use of organic solvents, and reduces the number of operating units.

[0005] Technical solution: To solve the above technical problems, the present invention provides the use of DESs in extracting or in situ separating statins from red yeast rice, wherein the DESs are Btac / Eg, Btac / Gly, Btac / Dim, Tebrc / La or Tebrc / Dim.

[0006] Wherein, the application also includes cellulase.

[0007] The application includes the following steps: adding a DESs enzyme system to red yeast rice powder, ultrasonic extraction, and centrifugation to extract or in situ separate the statin components; the DESs enzyme system consists of DESs, a H3C6H5O7 / Na3C6H5O7 buffer solution with a content of 20-70%, and cellulase.

[0008] Wherein, the molar ratio of Benzyltributylammonium chloride to Ethylene Glycol in the Btac / Eg is 1:2.

[0009] Wherein, the pH of the H3C6H5O7 / Na3C6H5O7 buffer solution is 3-6.

[0010] Furthermore, the pH of the H3C6H5O7 / Na3C6H5O7 buffer solution is 4-6.

[0011] Wherein, the content of the H3C6H5O7 / Na3C6H5O7 buffer solution is 20-80%.

[0012] Furthermore, the content of the H3C6H5O7 / Na3C6H5O7 buffer solution is 20-50%.

[0013] Furthermore, the content of the H3C6H5O7 / Na3C6H5O7 buffer solution is 20-40%.

[0014] Wherein, the ultrasonic extraction time is 5-60min.

[0015] Furthermore, the ultrasonic extraction time is 10-60 min.

[0016] Furthermore, the ultrasonic extraction time is 20-60 minutes.

[0017] Furthermore, the ultrasonic extraction time is 30-60 minutes.

[0018] Furthermore, the ultrasonic extraction time is 40-50 minutes.

[0019] Wherein, the temperature of the ultrasonic extraction is 25-65°C.

[0020] Furthermore, the temperature of the ultrasonic extraction is 25-55°C.

[0021] Furthermore, the temperature of the ultrasonic extraction is 35-55°C.

[0022] Furthermore, the temperature of the ultrasonic extraction is 35-45°C.

[0023] Wherein, the concentration of the cellulase is 0.5-2.0 mg / mL.

[0024] Furthermore, the concentration of the cellulase is 0.5-1.5 mg / mL.

[0025] Wherein, the liquid-to-solid ratio of the DESs enzyme system to red yeast rice powder is 10-60 mg / mL.

[0026] Furthermore, the liquid-to-solid ratio of the DESs enzyme system to red yeast rice powder is 30-60 mg / mL.

[0027] Furthermore, the liquid-to-solid ratio of the DESs enzyme system to red yeast rice powder is 40-60 mg / mL.

[0028] Wherein, the statin component includes one or more of lovastatin acid, lovastatin or deshydroxylovastatin.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. While establishing and optimizing a green extraction method for extracting and in-situ separating statins from red yeast rice, the present invention avoids the use of organic solvents, reduces the number of operating units, and has great application potential; 2. The enzyme system of the present invention is more compatible with cellulase than traditional organic solvents and has higher enzyme catalytic efficiency; 3. The strong solubility of DESs on substrates can overcome the limitation of poor solubility of traditional water-enzyme systems; 4. The present invention optimizes key process parameters and obtains the optimal process for extracting effective ingredients from red yeast rice based on the DESs system of BTAC / Eg (1:2). Under the optimal extraction conditions, the total extraction rate of effective ingredients is 9.209 mg / g, and its extraction efficiency is higher than that of conventional solvents (methanol, acetonitrile, ethanol and water); 5. The DESs solvent system of Btac / Eg (1:2) helps to promote the extraction and separation of effective ingredients from red yeast rice. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The ultra-high performance liquid chromatograms of red yeast rice extract (A) and mixed reference substance (B);

[0031] Figure 2 is the total extraction efficiency of DESs for lovastatin acid, lovastatin and deshydroxylovastatin in red yeast rice;

[0032] Figure 3 is the total extraction efficiency of the main statin components in red yeast rice by the DESs-based enzyme system;

[0033] Figure 4 is the infrared spectrum of Btac / Eg;

[0034] Figure 5For the extraction rate of LVSA, LVS, and DLVS: A. Optimization of extraction time; B. Optimization of extraction temperature; C. Optimization of liquid-solid ratio;

[0035] Figure 6 For the extraction rate of LVSA, LVS, and DLVS: A. Optimization of buffer content; B. Optimization of pH value of buffer solution; C. Optimization of enzyme concentration;

[0036] Figure 7 The response surface diagram of the factors affecting the extraction rate is as follows: A. extraction time and buffer pH; B. extraction time and buffer content; C. buffer pH and buffer content;

[0037] Figure 8 The extraction rates of LVSA, LVS and DLVS by Btac / Eg cellulose enzymatic hydrolysis system and traditional solvents;

[0038] Fig. 9 The phase diagram of Btac / Eg cellulose enzymatic hydrolysis system at different pH values;

[0039] Fig.10 Figures of red yeast rice powder after extraction with different solvents: A: red yeast rice powder; B: methanol extraction; C: ethanol extraction; D: Btac / Eg extraction. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0041] 1 Experimental Materials and Instruments

[0042] 1.1 Experimental instruments

[0043] The 1 / 100000 electronic analytical balance was purchased from Sartorius Group (Beijing, China); the 1 / 1000000 electronic balance and electronic analytical balance were purchased from Tianma Hengji Instrument Co., Ltd. (Tianjin, China); the KH-500DB digital ultrasonic cleaner was purchased from Kunshan Hechuang Ultrasonic Instrument Co., Ltd. (Suzhou, China); Agilent 1260infinity II high performance liquid chromatograph and Aglient 1290UHPLC-6530QTOF MS were purchased from Santa Clara, USA; the centrifuge was purchased from Hunan Xiangyi Centrifuge Instrument Co., Ltd. (Changsha, China); the pipette was purchased from Eppendorf (Hamburg, Germany); the 0.45 μm organic filter was purchased from Tianjin Linghang Experimental Equipment Co., Ltd. (Tianjin, China), and the HJ-4A multi-head magnetic heating stirrer was purchased from Guohua Electric Co., Ltd. (Changzhou, China).

[0044] 1.2 Experimental reagents and drugs

[0045] Chromatographically pure acetonitrile was purchased from Merck (Darmstadt, Germany); chromatographically pure formic acid was purchased from ROE (Newark, USA); analytically pure methanol, anhydrous ethanol, and acetonitrile were all purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China); deionized water (18MΩ·cm) was purified by Milli-Q water system (Millipore, Massachusetts, USA). Red yeast rice was purchased from the Affiliated Hospital of Yangzhou University and identified as the dried root of Aspergillus fungi by Professor Liu Liang of Yangzhou University. The red yeast rice was crushed and passed through a 50-mesh sieve. The resulting powder was placed in a brown glass bottle and stored in a refrigerator at 4°C. Lovastatin (Lovastatin, LVT, purity>98%), lovastatin acid (Lovastatin acid, LVTA, purity>98%), and dehydrolovastatin (Dehydrolovastatin, DLVT, purity>98%) were all purchased from Chengdu Pufeide Reference Material Technology Co., Ltd. The compound structure is as follows:

[0046]

[0047] Phenyltributylammonium chloride (Btac, >97%) and triethylbenzylammonium chloride (Tebrc, >98%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China). Hexadecyltrimethylammonium chloride (Ctac, >98%), tetrabutylammonium chloride (Tbac, >96%), tetraethylammonium chloride (Teac, >98%), tetramethylammonium chloride (Tmac, >98%), tetrapropylammonium chloride (Tpac, >96%), malonic acid (MDA, >98%), urea (Ure, >98%), xylitol (Xyl, >98%), ethylene glycol (Eg, >98%), glycerol (Gly, >96%), levulinic acid (La, >98%), L-malic acid (L-Ma, >98%), D-sorbitol (D-Sor, >97%), N,N-dimethylurea (Dim, >98%), acetamide (Ac, >98%), citric acid monohydrate (>98%) and sodium citrate dihydrate (>98%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China).

[0048] 2. Chromatographic conditions

[0049] Agilent 1260infinity II system was used, which was equipped with a binary pump, VWD detector, and column oven. The chromatographic column was Agilent InfinityLab Poroshell 120EC-C18 (4.6mm×250mm, 5μm), the mobile phase was acetonitrile (B)-0.1% (v / v) phosphoric acid aqueous solution (D), and the elution gradient was: 0-42min, 25-84% B; 42-46min, 84-100% B. The flow rate was 1mL / min, the injection volume was 5μL, the column temperature was 30°C, and the detection wavelength was 273nm.

[0050] 3. Sample solution preparation

[0051] Weigh 25 mg of red yeast rice powder accurately and place it in a conical flask. Add 1 mL of methanol and perform ultrasonic extraction (40 KHz, 500 W) for 60 min at room temperature. Centrifuge the extract at 13500 rcf for 10 min, filter it through a 0.22 μm filter membrane, and place the supernatant in a sample vial for sample analysis. The results are shown in Table 1. Figure 1 As shown, it shows that the three target analytes are clearly separated by chromatography in the red yeast rice sample and can be used for subsequent quantitative analysis. In the figure, 1 is lovastatin acid; 2 is lovastatin; and 3 is deshydroxylovastatin.

[0052] Example 1 Methodological Investigation

[0053] 1. Linearity, limit of quantification (LOQ) and limit of detection (LOD)

[0054] Three compound reference substances (lovastatin, lovastatin acid, deshydroxylovastatin) were accurately weighed and prepared with methanol solution to obtain a mixed reference substance mother solution containing 2102 μg of lovastatin acid, 1674 μg of lovastatin, and 128 μg of deshydroxylovastatin per 1 mL. The mother solution was then diluted with methanol to form a series of gradient concentration solutions (the concentration of lovastatin acid was 105.1 ug / mL-2102.00 ug / mL; the concentration of lovastatin was 83.7 ug / mL-1674.00 ug / mL; the concentration of deshydroxylovastatin was 6.40 ug / mL-128.00 ug / mL), and then liquid phase analysis was performed according to the above-mentioned chromatographic conditions. The concentration was linearly regressed using the peak area of ​​the test compound to obtain the standard curve of each compound. The reference solution of the above concentration was diluted step by step in equal proportions and then analyzed by liquid phase according to the above chromatographic conditions, and the concentrations when the signal-to-noise ratio (S / N) was 10 and 3 were used as the quantitative limit and the detection limit, respectively. The results are shown in Table 1.

[0055] 2. Precision inspection

[0056] Intra-day precision: Take any reference solution under "Linearity, limit of quantitation (LOQ) and limit of detection (LOD)" (the concentrations of the reference solution used in this example are: lovastatin acid is 420.4ug / mL; lovastatin is 334.8ug / mL; lovastatin acid is 25.6ug / mL), inject it 6 times in one day, record the retention time and peak area of ​​the target compound, and calculate the relative standard deviation (RSD).

[0057] Inter-day precision: The above-mentioned reference solution was injected three times every day for three consecutive days, the retention time and peak area of ​​each compound were recorded, and the relative standard deviation (RSD) was calculated.

[0058] As shown in Table 1, the RSD of the intra-day precision of the three analytes was 0.95%-2.31%, and the RSD of the inter-day precision was 1.80%-3.92%, both less than 5%, indicating that the intra-day precision and inter-day precision of this method were good.

[0059] 3. Repeatability test

[0060] According to the above sample preparation method, 6 red yeast rice sample solutions were prepared in parallel. The 6 samples were subjected to liquid phase analysis according to the above chromatographic conditions, and the percentage of each compound was calculated and the RSD value of the determination results was calculated. The results show (Table 1) that the RSD values ​​(Repeatability) of the target compound content in red yeast rice are all less than 2.85%, indicating that the repeatability of this method is good.

[0061] 4. Stability inspection

[0062] The samples prepared according to the above sample preparation method were subjected to liquid phase analysis according to the above chromatographic conditions at 0h, 2h, 4h, 6h, 8h, 12h and 24h, respectively, and the peak area of ​​each compound was recorded, and the RSD value was calculated to examine the stability of the sample. The results show (Table 1) that the RSD value (Stability) of the peak area of ​​the target compound in red yeast rice is less than 3.39%, indicating that the sample solution is stable within 24h.

[0063] 5. Accuracy test

[0064] The accuracy was investigated by the method of sample recovery: a certain amount of red yeast powder was accurately weighed, and 6 portions were paralleled, and then reference substances equivalent to the content of the components to be tested in the samples were added respectively, and the samples were prepared according to the above sample preparation method, and liquid phase analysis was performed according to the above chromatographic conditions, and the sample recovery and RSD value of each compound were calculated. The calculation formula of the sample recovery is: recovery rate (%) = (actual measured amount - medicinal material content) / reference substance added amount × 100%. The results are shown in Table 1. The average recovery rate of the components to be tested in red yeast was 95.56%-104.72%, and the RSD value was 3.20%-4.93%, which met the requirements of the pharmacopoeia, indicating that the method has good accuracy.

[0065] Table 1 Standard curves and methodological investigation results of the components to be tested in red yeast rice

[0066]

[0067] Example 2 Preparation and screening of DESs

[0068] 1. Preparation of DESs

[0069] First, 58 kinds of DESs were synthesized according to Table 2. According to the specific molar ratio, appropriate amounts of HBD and HBA were weighed in the same beaker, heated to 50-80°C, and stirred continuously at this temperature until a uniform, clear liquid was formed. After cooling to room temperature, the mixture was stored in a desiccator for later use.

[0070] Table 2 58 DESs based on polyalkyl substituted ammonium chloride

[0071]

[0072]

[0073]

[0074] 2. DES screening

[0075] First, a series of DESs-based solvent systems (1-58) were prepared. The DESs-based solvent system consisted of 70% DESs (v / v) and 30% water (v / v). 50 mg of red yeast rice powder was accurately weighed, 1 mL of the prepared solvent system was added, and ultrasonic (40 KHz, 500 W, 35 ° C) extraction was performed for 60 min. The extract was centrifuged at 13000 rpm for 10 min, and the supernatant was diluted 5 times with pure methanol and filtered with a 0.22 μm microporous filter membrane for sampling and analysis. Based on the total extraction rate of lovastatin acid, lovastatin and deshydroxylovastatin (active ingredients) in red yeast rice, a DES solvent system with a total extraction rate significantly stronger than methanol was preliminarily screened for optimal enzyme system screening. The results are shown in Tables 3 and Figure 2 shown.

[0076] Table 3 Contents of three compounds in 58 DESs systems (mg / g)

[0077]

[0078]

[0079] like Figure 2 As shown in the figure, 58 DESs solvent systems were screened, and compared with methanol extraction, it was found that a total of 11 DESs solvent systems could effectively improve the total extraction rate of lovastatin acid, lovastatin and deshydroxylovastatin (6.44-8.03 mg / g). Overall, the DESs solvent system with Btac and Tbac as hydrogen bond acceptors and Eg as hydrogen bond donor can effectively improve the extraction rate of statins in red yeast rice. The total extraction rates of these three active ingredients in different DESs systems are ranked from high to low as follows: Btac / Eg (1:2) > Btac / Dim (1:2) > Tbac / Eg (1:2) > Tpac / La (1:2) > Btac / Gly (1:2) > Tebrc / La (1:2) > Tebrc / Dim (1:2) > Btac / Xyl (1:2) > Tbac / Ac (1:2) > Btac / La (1:2) > Tmac / Ac (1:2).

[0080] 3. Screening of optimal enzyme system based on DESs

[0081] According to the results of the initial screening of DESs, a DES solvent system with a total extraction rate significantly higher than that of methanol was selected to prepare a series of enzyme systems based on DESs. The enzyme system based on DESs consists of 70% DESs (v / v), 30% citric acid and sodium citrate pentahydrate buffer solution (v / v, pH 5, the volume ratio of H3C6H5O7 / Na3C6H5O7 is 8.2:11.2) and cellulase (1 mg / mL). Among them, DESs are Btac / Xyl, Btac / Eg, Btac / Gly, Btac / La, Btac / Dim, Tbac / Eg, Tbac / Ac, Tmac / Ac, Tpac / La, Tebrc / La or Tebrc / Dim. 50 mg of red yeast rice powder was accurately weighed, 1 mL of the prepared solvent system was added, and ultrasonic (40 KHz, 400 W, 35 ° C) extraction was performed for 60 min. The extract was centrifuged at 13000 rpm for 10 min, and the supernatant was diluted to 5 times and filtered through a 0.22 μm microporous filter for analysis. The optimal enzyme system based on DESs was screened based on the total extraction rate of lovastatin acid, lovastatin and deshydroxylovastatin (active ingredients) in red yeast rice. Figure 3As shown in the figure, among the 11 DESs tested, the addition of cellulase to Btac / Xyl, Btac / La, Tbac / Eg, Tbac / Ac, Tmac / Ac and Tebrc / La reduced their extraction rates of the target compounds. For the remaining five DESs, the addition of cellulase significantly increased the extraction rates of the target compounds. Among them, the extraction rate of statins by the Btac / Eg cellulase system was significantly higher than that of other DESs enzyme systems. For the cellulase system and statins in red yeast rice, the Btac / Eg cellulase system had the best extraction efficiency, so the Btac / Eg cellulase system was selected for further experiments.

[0082] 4. Characterization of Optimal DSE

[0083] Fourier transform infrared spectrometer at 4000-550 cm -1 The spectrum of the best DES system was evaluated in the wavenumber range, and the structure of the synthesized DES was characterized. Figure 4 As shown in FIG. 1 , a Fourier transform infrared spectrometer is used to detect the infrared spectra of Btac, Eg and Btac / Eg. For Btac, the infrared spectrum is between 3200 and 3400 cm -1 There is a broad peak between 2900 cm -1 and 2850cm -1 The peak at 3400 to 3600 cm-1 corresponds to the stretching vibration of the aliphatic CH bond in the benzyl group. -1 The broad peak between 1100 cm -1 The characteristic peaks near 900cm -1 The peaks near 3400 to 3300 cm-1 are related to the stretching vibration of the CC bond in Eg. -1 The characteristic peaks were blue-shifted, indicating that the OH group of Eg interacted with the NH group of Btac. -1 The red shift of the nearby characteristic peaks further confirmed the formation of hydrogen bonds between the OH of Eg and the NH of Btac.

[0084] Example 3 Optimization of the extraction process of Btac / Eg cellulose enzymatic hydrolysis system based on a single factor

[0085] 1. Univariate analysis

[0086] The key parameters in the extraction process of the optimal enzyme system of DESs were optimized by single factor experimental design, including liquid-solid ratio, enzyme concentration, H3C6H5O7 / Na3C6H5O7 buffer content, buffer solution pH value, extraction time and extraction temperature. The detailed optimization experimental design is shown in Table 4. After extraction, centrifugation was performed at 13500rpm for 10min, the supernatant was diluted to an appropriate concentration, and filtered with a 0.22μm microporous filter membrane for sample analysis. Subsequently, the total extraction rate of lovastatin acid, lovastatin and deshydroxylovastatin in red yeast rice was used as an indicator, and the key extraction parameters were optimized by single factor experimental design to determine the optimal extraction conditions.

[0087] Table 4 Single factor optimization experimental design

[0088]

[0089]

[0090] The effect of extraction temperature on extraction efficiency is as follows Figure 5 As shown in Figure B. When the extraction temperature increases from 25°C to 45°C, the extraction rate of the target compound increases significantly. When the temperature continues to rise to 55°C, the extraction rate of the target compound does not change much. From 55°C to 65°C, the extraction rate of the target compound decreases. This is because the enzyme can only maintain catalytic activity within an appropriate temperature range. When the temperature is below 45°C, as the temperature increases, the movement of the molecules increases, making it easy for the substrate to bind to the enzyme, promoting the enzymatic reaction, and ultimately increasing the extraction rate of the target compound. However, when the temperature is too high, the enzyme will gradually lose its activity, resulting in a decrease in the extraction rate. Therefore, under mild conditions, 45°C is the best choice to ensure extraction efficiency.

[0091] The effect of extraction time on the extraction rate is as follows: Figure 5 As shown in A. When the extraction time increased from 5 min to 40 min, the extraction rate of the target compound increased significantly, but as the extraction time further increased, the extraction rate of the target compound did not change significantly. Therefore, the optimal extraction time was 40 min.

[0092] The effect of liquid-solid ratio on the extraction rate is as follows Figure 5 C. When the liquid-solid ratio increased from 5:1 mg / mL to 50:1 mg / mL, the extraction rate of the target compound increased significantly. When the liquid-solid ratio continued to increase from 50:1 mg / mL to 60:1 mg / mL, the extraction rate of the target compound did not increase significantly. The effect of the liquid-solid ratio on the extraction effect is achieved by affecting the solubility and concentration gradient of the target compound. Considering the amount of solvent used, 50:1 mg / mL was selected as the optimal liquid-solid ratio.

[0093] The effect of buffer content on the extraction efficiency is shown in Figure 6A. When the buffer content increased from 10% (v / v) to 30% (v / v), the extraction rate of statins increased significantly. As the buffer content continued to increase from 30% (v / v) to 80% (v / v), the extraction rate of statins decreased significantly. On the one hand, DESs can enhance the dissolution of substrates and the destruction of cell walls, thereby enhancing extraction and conversion. On the other hand, the viscosity of DESs will hinder the movement of substrate molecules, which is not conducive to the binding and diffusion of enzymes and substrates. The higher the DESs content in the system, the higher the viscosity of the system, which may reduce extraction and conversion. However, when the DESs content in the system decreases, its dissolution of the target components and destruction of the cell walls are weaker, which is not conducive to extraction and conversion. The ultimate effect of DESs on the extraction and conversion process depends on the combined effect of these two factors. The results show that the optimal buffer content is 30% (v / v).

[0094] Effect of pH value of buffer solution on extraction efficiency Figure 6 As shown in Figure B. When the pH value increases from 3 to 5, the extraction rate of the target compound also increases significantly. When the pH increases from 5 to 6, the extraction rate decreases. The activity of the enzyme requires an appropriate pH range. Under highly acidic and alkaline conditions, the structure of the enzyme will be destroyed and the enzyme will be inactivated. The decrease in enzyme activity leads to a decrease in the ability to destroy the cell wall, which in turn leads to a decrease in the extraction rate. The results show that when the buffer pH value is 5, the enzyme activity is the highest and the extraction effect of the target compound is the best.

[0095] Effect of cellulase concentration on extraction efficiency Figure 6 C. When the cellulase concentration increased from 0.5 mg / mL to 1 mg / mL, the extraction rate of the target compound also increased. However, when the enzyme concentration was further increased to 2.0 mg / mL, the extraction rate decreased. This may be because the viscosity of the DES system is relatively high, and the addition of the enzyme further increases its viscosity, hindering the movement of molecules and reducing the binding and mass transfer process between the enzyme and the substrate. Therefore, the optimal concentration of cellulase is 1 mg / mL.

[0096] The optimal extraction conditions based on single-factor optimization experimental conditions were: extraction temperature 45°C, buffer solution pH 5, cellulase concentration 1 mg / mL, buffer solution content 30% (v / v), liquid-solid ratio 50:1 mg / ml, and ultrasonic extraction time 40 min.

[0097] 2. Response surface analysis

[0098] The single factor experiment showed that DESs extraction time, buffer pH value and buffer content had a great influence on the extraction rate of statins in red yeast rice. Subsequently, the Box-Behnken experimental design principle was used to select three factors and three levels for optimization research. The three factors were recorded as A (buffer content%), B (buffer pH), and C (extraction time). The high, medium, and low levels of each factor were recorded as 1, 0, and -1, respectively. The levels of each factor are shown in Table 5. The total content of TTSG, ED, and EME in red yeast rice was used as the response value, and the Box-Behnken experimental design established by Design Expert software was used for response surface analysis.

[0099] Table 5 Box-Behnken experimental design table

[0100]

[0101] The extraction rates of lovastatin acid, lovastatin and deshydroxylovastatin determined under 17 different conditions are shown in Table 6.

[0102] Table 6 Extraction rates of lovastatin acid, lovastatin and deshydroxylovastatin under different conditions

[0103]

[0104]

[0105] Note: A, extraction time (min); B, pH value; C, buffer content (%)

[0106] 3. Establishment of quadratic regression model

[0107] The experimental results in Table 6 were subjected to multivariate linear regression and binomial fitting, and the mathematical simulation of the total extraction rate of lovastatin acid, lovastatin, and deshydroxylovastatin in red yeast rice was as follows:

[0108] Y=0.967+0.020×A-0.011×B-0.038×C+0.004×AB-0.007×AC+0.050×BC-0.018×A 2 -0.053×B 2 -0.066×C 2

[0109] Wherein Y represents the extraction rate (mg / g), A, B, and C represent the extraction time, pH of the buffer, and the content of the buffer, respectively.

[0110] Table 7 Analysis of variance table of regression equation

[0111]

[0112]

[0113] The above regression equation was subjected to variance analysis, and the results are shown in Table 7. When P<0.05, the effect of the factor on the response value is significantly different, and when P<0.001, it is an extremely significant difference. The model of this experiment is significant (P<0.001), indicating that the regression model indicators are highly significant; the lack of fit term (Lack of Fit) is used to evaluate the model fit. If it is not significant, it indicates that the equation simulation is good. The results show that the model lack of fit term P=0.2577>0.05, indicating that the model fit is good; the correlation coefficient R 2 =0.9998, which further shows that the model has good credibility.

[0114] The analysis results of red yeast rice extraction conditions are as follows: Figure 7 As shown. The extraction time, buffer pH and buffer content all had significant effects on the extraction of statins (P<0.05). The extraction efficiency of statins continued to increase until the extraction time (variable A) reached 45 minutes; when the extraction time exceeded 45 minutes, the extraction efficiency of statins was negatively correlated with it. As the extraction time decreased, the extraction efficiency of statins increased. The extraction efficiency of statins continued to increase until the buffer pH (variable B) reached 5.2; when the pH was greater than 5.2, the extraction efficiency of statins was negatively correlated with it. As the pH decreased, the extraction efficiency of statins increased. The extraction efficiency of statins continued to increase until the buffer content (variable C) reached 31.5%; when the buffer content was greater than 31.5%, the extraction efficiency of statins was negatively correlated with it. As the buffer content decreased, the extraction efficiency of statins increased. The extraction time (variable A) and the extraction efficiency were positively correlated, but the trend was not obvious (P=0.0723).

[0115] The extraction method was optimized by Design Expert software, and the optimized extraction conditions were as follows: the water content in DES was 31.5%, the pH of the buffer was 5.26, and the extraction time was 44 min. Under this optimized condition, the predicted value of alkaloid extraction efficiency was 9.138 mg / g. The alkaloids in Tripterygium wilfordii were extracted according to the above optimized conditions for experimental verification. The average result of the three extraction experiments was 9.209 mg / g, which was close to the theoretical value, indicating that this model is reliable. The research results show that response surface analysis can effectively predict the optimal extraction conditions, can be applied to the optimization of extraction conditions of active ingredients in traditional Chinese medicine, and has good application prospects.

[0116] Example 4 Comparison of the extraction efficiency of Btac / Eg cellulose enzymatic hydrolysis system and traditional solvent system

[0117] Although the Btac / Eg cellulose enzymatic hydrolysis system has the highest extraction rate of the target compound, it is still unknown whether the DESs-based cellulose enzyme system is superior to water and organic solvents and traditional solvent enzyme systems. Therefore, this example compares the extraction efficiency of the Btac / Eg cellulose enzymatic hydrolysis system under the optimal extraction conditions with the conventional solvent system under the same conditions.

[0118] Table 8 Extraction rate of LVSA, LVS and DLVS in red yeast rice by different solvent systems (mg / g)

[0119]

[0120] The results are as follows Figure 8 As shown in Table 8, compared with conventional extraction solvents (methanol, ethanol, acetonitrile and water), the extraction rate of LVSA, LVS and DLVS in red yeast rice by Btac / Eg cellulase system is significantly higher than that of conventional extraction solvents, and the extraction rate is as follows: Btac / Eg cellulase system (9.202 mg / mL) > methanol (6.123 mg / mL) > ethanol (4.779 mg / mL) > acetonitrile (4.220 mg / mL) > water (3.897 mg / mL). The extraction rate of LVSA, LVS and DLVS in red yeast rice by Btac / Eg cellulase system is 1.5 times that of methanol extraction, indicating that the extraction efficiency of Btac / Eg cellulase system is significantly stronger than that of conventional extraction solvents.

[0121] Example 5 Reversible phase behavior of the optimal DES enzyme system controlled by pH and its effect on the in situ separation process

[0122] 1. Reversible phase behavior of the optimal DES enzyme system controlled by pH

[0123] The previous experimental results show that the Btac / Eg cellulose enzymatic hydrolysis system is indeed a system with higher efficiency than the traditional solvent system. In order to achieve in situ separation of the target compound, this example studies whether the Btac / Eg cellulose enzymatic hydrolysis system has a reversible phase behavior controlled by pH value.

[0124] First, prepare a H3C6H5O7 / Na3C6H5O7 buffer solution with a pH value of 5 and a concentration of 1.395 mol / L. Then divide the prepared buffer solution into three parts, add appropriate amounts of sodium hydroxide or hydrochloric acid solution to adjust the pH value to 3.13, 4.76 or 6.40 respectively. Add 2 mL of the optimal DES enzyme system solution containing a cellulase concentration of 1.5 mg / mL into three glass tubes respectively. Use a burette to drop the prepared H3C6H5O7 / Na3C6H5O7 buffer solutions with pH values ​​of 3.13, 4.76, and 6.40 in sequence. When the solution in each tube is just turbid, record the consumption of the buffer solution. Then add 0.2 mL of water to make each solution clear again. Repeat the above operation 7 times. Calculate the concentration (w / v) of the optimal DES enzyme system and H3C6H5O7 / Na3C6H5O7 in the system when turbidity occurs, and draw a phase diagram. All the above experiments were carried out at 20°C. According to the phase diagram, the optimal DES enzyme system with specificity was prepared. The pH value of these systems was continuously adjusted to observe whether phase separation occurred and whether the phase separation was reversible. The effect of phase behavior on the separation effect was studied by calculating the distribution of the target compound in the two phases at different pH and different system compositions.

[0125] When pH < 3.13, the buffer exists in the form of H3C6H5O7, and its salting-out ability is weak. It can be seen that the Btac / Eg cellulose hydrolysis system does not form a two-phase aqueous system when the pH is lower than 3.13. When the pH is between 3.13-4.76, or continues to increase to 6.4, the buffer in the system exists in the form of NaH2C6H5O7 or Na2HC6H5O7, and the salting-out ability is enhanced. When the pH continues to increase (> 6.4), the system exists in the form of Na3C6H5O7, which has the strongest salting-out ability and is most likely to form two aqueous phases (the upper phase is DESs and the lower phase is the salt phase). Therefore, by controlling the pH value, the reversible transformation of single phase and two phases can be successfully achieved. Fig. 9 As shown, at pH 5.2 and pH 6.6, a two-phase aqueous system was formed. Specifically, when the composition of the Btac / Eg cellulose enzymatic hydrolysis system was between the red line and the black line, the transition between the single phase and the two phases could be achieved by controlling the pH value between 5.2 and 6.6. Therefore, the dynamic change of H3C6H5O7 / Na3C6H5O7 in the Btac / Eg cellulose enzymatic hydrolysis system can be achieved by adjusting the pH, and a pH-controllable DESs-based reversible enzyme system was developed.

[0126] 2. In situ separation of target compounds by controlling the pH value and composition of the Btac / Eg cellulose enzymatic hydrolysis system

[0127] Although the Btac / Eg cellulose enzymatic hydrolysis system has a pH-controlled reversible phase behavior, whether the target compounds can be separated needs further study. Therefore, five points with a large coverage of component differences were selected in the phase diagram, such as Fig.10 As shown in the figure, the transition between single phase and two phases was achieved by adjusting the pH value and composition of the Btac / Eg cellulose enzymatic hydrolysis system at room temperature. The separation effect was evaluated by examining the content of each component in the two-phase extract. The results showed that with the increase of H3C6H5O7 / Na3C6H5O7 concentration and pH value in the Btac / Eg cellulose enzymatic hydrolysis system, the isoflavone content in the Btac / Eg cellulose enzymatic hydrolysis system increased, and the enrichment efficiency of statins at 5 points was greater than 90%. In particular, under the combination of point 3, the separation effect was the best, and almost all (>97%) of the target statins were in the upper phase enriched by the Btac / Eg cellulose enzymatic hydrolysis system, thus achieving the in-situ separation of statins in red yeast rice.

[0128] 3. Scanning electron microscope analysis results

[0129] Broken plant cells can promote the dissolution of target components from plants and lead to relatively high extraction rates. Therefore, the morphology of red yeast rice powder after extraction with Btac / Eg cellulose enzymatic hydrolysis system and other commonly used extraction solvents (methanol and ethanol) was observed using scanning electron microscopy. Fig.10 As shown in A, before extraction, it was observed that the red yeast rice powder had a complete cell wall structure and the cells were closely arranged. After various extraction processes, the surface of the red yeast rice powder became rough and obvious damage appeared on the surface ( Fig.10 B, 10C and 10D). The degree of cell wall damage was in the order of Btac / Eg cellulose enzymatic hydrolysis system > methanol > ethanol. The results of scanning electron microscopy partly explained that the Btac / Eg cellulose enzymatic hydrolysis system had a better extraction efficiency for statins in red yeast rice.

Claims

1. The use of DESs in extracting, in-situ separating or recovering statins from red yeast rice, characterized in that: The DESs are Btac / Eg, Btac / Gly, Btac / Dim, Tebrc / La or Tebrc / Dim.

2. The application according to claim 1, characterized in that: The application also includes cellulase.

3. The use according to claim 1 or 2, characterized in that: The application comprises the following steps: adding a DESs enzyme system to red yeast rice powder, ultrasonic extraction, and centrifugation to extract or in situ separate the statin components; the DESs enzyme system is composed of DESs, a H3C6H5O7 / Na3C6H5O7 buffer solution with a content of 20-70%, and cellulase.

4. The use according to claim 3, characterized in that: The pH of the H3C6H5O7 / Na3C6H5O7 buffer solution is 3-6.

5. The application according to claim 3, characterized in that: The content of the H3C6H5O7 / Na3C6H5O7 buffer solution is 20-50%.

6. The use according to claim 3, characterized in that: The temperature of the ultrasonic extraction is 25-65°C.

7. The use according to claim 3, characterized in that: The ultrasonic extraction time is 5-60min.

8. The use according to claim 3, characterized in that: The concentration of the cellulase is 0.5-2.0%.

9. The use according to claim 3, characterized in that: The liquid-to-solid ratio of the DESs enzyme system to red yeast rice powder is 10-60 mg / mL.

10. The use according to claim 1 or 2, characterized in that: The statin component includes one or more of lovastatin acid, lovastatin or deshydroxylovastatin.

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